丙型肝炎病毒NS2蛋白在Huh-7细胞中抑制NF-κB活性

Hepatitis C Virus NS2 Protein Suppresses the Activities of NF-κB in Huh-7 Cell Line

将丙型肝炎病毒(HCV)非结构蛋白NS2基因的全长序列插入到真核表达载体pCDNA3.1(-)CMV启动子下游,构建成重组质粒pCNS2.用脂质体LipoVecTM转染Huh-7细胞,转染细胞内可检出NS2的mRNA和蛋白质,表明构建的pCNS2可在Huh-7细胞内成功表达;把不同剂量的pCNS2质粒DNA与报告质粒pNF-κ B-Luc共转染Huh-7细胞,48h后检测荧光素酶活性,结果显示与pCNS2共转染的细胞中pNF-κ B-Luc表达出的荧光素酶的活性比对照细胞降低了约2～4倍,并呈明显的剂量相关性.表明HCV NS2对NF-κ B激活转录活性有明显的抑制作用.这可能与HCV慢性持续性感染的致病性有一定的相关性.

Recombinant plasmid pCNS2 was constructed by inserting Hepatitis C Virus full-length NS2 gene between Xho I and Hind III sites of an eukaryotic expression vector pCDNA3.1(-). The recombin- ant plasmid pCNS2 was transfected to Huh-7 cells with LipoVecTM and HCV NS2 mRNA and protein produced in transient expression system was detected at the transcriptional and translational levels, respe- ctively. This indicated that pCNS2 could be expressed successfully in Huh-7 cells. Cotransfected pCNS2 of different doses to Huh-7 cells with reporter plasmid pNF-κB-Luc,the activity of luciferase controlled by NF-κB promoter/enhancer elements was detected by luciferase assay kit 48h after transfection. The results showed that the expression of luciferase in Huh-7 cells transfected by the pCNS2 decreased about two to four folds compared to control plasmid. It is suggested that the HCV NS2 protein distinctly suppressed the activities of NF-κB in a dose-dependent manner in Huh-7 cells and this will enable to investigate on the role of NS2 in the pathogenesis of chronic HCV infection.